Infection by human immunodeficiency virus (HIV) is initiated by fusion between the viral envelope and plasma membrane. Fusion is mediated by the envelope protein (Env), which undergoes conformational changes by sequential binding to receptor (CD4) and coreceptor (chemokine receptors), culminating in a sixhelix bundle structure (6HB). Conserved sites of Env are exposed during the refolding and this provides an attractive target for therapeutic intervention. Env-derived peptides potently inhibit HIV entry by binding to Env in a pre-bundle conformation and preventing its folding into a 6HB. One of these peptides is now used to treat AIDS patients. Understanding the molecular mechanism of Env-induced fusion will allow for design of anti-viral strategies. A powerful means to study the HIV entry mechanism is to capture and characterize key intermediate stages of fusion. HIV Env will be expressed in effector cells and fused to target cells expressing CD4 and coreceptors. Fusion will be monitored by fluorescence microscopy, real-time electrical admittance measurements and flow cytometry. The progression of fusion through its intermediate stages will be evaluated by measuring, at each intermediate, the potency of inhibitors that target specific sites of coreceptors and Env. The mechanism of HIV resistance to 6HB-blocking peptides will be investigated by arresting fusion at intermediates at which the peptide binding sites are stably exposed. Increased potency of these peptides added at an intermediate stage would imply that, under normal conditions, the longevity of exposure of these sites determines the sensitivity to peptides. To capture the stage at which Env inserts its hydrophobic segment (fusion peptide) into the target membrane, a 6HB-blocking peptide anchored to the target membrane will be used. An important question is whether the non-covalently associated surface and transmembrane subunits of Env must disengage during fusion. This will be addressed by identifying the intermediate stage that is captured by linking the subunits together, and changes that occur upon breaking the linkage. These studies will both advance our understanding of the mechanism by which HIV Env promotes virus entry and aid the development of novel entry inhibitors.